Gene for Identifying Individuals with Familial Dysautonomia

ABSTRACT

This invention relates to methods and compositions useful for detecting mutations which cause Familial Dysautonomia. Familial dysautonomia (FD; Riley-Day syndrome), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we mapped the FD gene, DYS, to a 0.5 cM region of chromosome 9q31 and showed that the ethnic bias is due to a founder effect, with &gt;99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its 5 genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA from FD patients, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from patient lymphoblasts is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19 that is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

This application is a continuation under 35 U.S.C. §120 of applicationSer. No. 12/172,847, filed Jul. 14, 2008, which is a continuation ofapplication Ser. No. 10/041,856, filed Jan. 7, 2002, now U.S. Pat. No.7,388,093, which claims priority to U.S. provisional application No.60/260,080, filed on Jan. 6, 2001, the contents of each of which areincorporated herein in their entirety.

This invention was made with government support under Grant NumberNS36326 awarded by The National Institutes of Health. The U.S.government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the gene, and mutations thereto,that are responsible for the disease familial dysautonomia (FD). Moreparticularly, the invention relates to the identification, isolation andcloning of the DNA sequence corresponding to the normal and mutant FDgenes, as well as characterization of their transcripts and geneproducts. This invention also relates to genetic screening methods andkits for identifying FD mutant and wild-type alleles, and furtherrelates to FD diagnosis, prenatal screening and diagnosis, and therapiesof FD, including gene therapeutics and protein/antibody basedtherapeutics.

BACKGROUND OF THE INVENTION

Familial Dysautonomia (FD, Riley-Day Syndrome, Hereditary Sensory andAutonomic Neuropathy Type III) [OMIM 223900] is an autosomal recessivedisorder present in 1 in 3,600 live births in the Ashkenazi Jewishpopulation. This debilitating disorder is due to the poor development,survival, and progressive degeneration of the sensory and autonomicnervous system (Axelrod et al., 1974). FD was first described in 1949based on five children who presented with defective lacrimation,excessive sweating, skin blotching, and hypertension (Riley et al.,1949). The following cardinal criteria have evolved for diagnosis of FD:absence of fungiform papillae on the tongue, absence of flare afterinjection of intradermal histamine, decreased or absent deep tendonreflexes, absence of overflow emotional tears, and Ashkenazi Jewishdescent (Axelrod and Pearson, 1984, Axelrod 1984).

The loss of neuronal function in FD has many repercussions, withpatients displaying gastrointestinal dysfunction, abnormal respiratoryresponses to hypoxic and hypercarbie states, scoliosis, gastroesophagealreflux, vomiting crises, lack of overflow tears, inappropriate sweating,and postural hypotension (Riley et al. 1949; Axelrod et al. 1974,Axelrod 1996). Despite recent advances in the management of FD, thedisorder is inevitably fatal with only 50% of patients reaching 30 yearsof age. The clinical features of FD are due to a genetic defect thatcauses a striking, progressive depletion of unmyelinated sensory andautonomic neurons (Pearson and Pytel 1978a; Pearson and Pytel 1978b;Pearson et al. 1978; Axelrod 1995). This neuronal deficiency beginsduring development, as extensive pathology is evident even in theyoungest subjects. Fetal development and postnatal maintenance of dorsalroot ganglion (DRG) neurons is abnormal, significantly decreasing theirnumbers and resulting in DRG of grossly reduced. size. Slow progressivedegeneration is evidenced by continued neuronal depletion withincreasing age. In the autonomic nervous system, superior cervicalsympathetic ganglia are also reduced in size due to a severe decrease inthe neuronal population.

Previously, the FD gene, DYS, was mapped to an 11-cM region ofchromosome 9q31 (Blumenfeld et al. 1993) which was then narrowed byhaplotype analysis to <0.5 cM or 471 kb (Blumenfeld et al. 1999). Thereis a single major haplotype that accounts for >99.5% of all FDchromosomes in the Ashkenazi Jewish (AJ) population. The recentidentification of several single nucleotide polymorphisms (SNPs) in thecandidate interval has allowed for further reduction of the candidateregion to 177 kb by revealing a common core haplotype shared by themajor and one previously described minor haplotype (Blumenfeld et al.1999).

SUMMARY OF THE INVENTION

This invention relates to mutations in the IKBKAP gene which theinventors of this invention discovered and found to be associated withFamilial Dysautonomia. The mutation associated with the major haplotypeof FD is a base pair mutation, wherein the thymine nucleotide located atby 6 of intron 20 in the IKBKAP gene is replaced with a cytosinenucleotide (T→C) (hereinafter “FD 1 mutation”). The mutation associatedwith the minor haplotype is a base pair mutation wherein the guaninenucleotide at by 2397 (bp 73 of exon 19) is replaced with a cysteinenucleotide (G→C) (hereinafter “FD2 mutation”). This base pair mutationcauses an arginine to proline missense mutation (R696P) in the aminoacid sequence of the IKBKAP gene that is predicted to disrupt apotential phosphorylation site.

In accordance with one aspect of the present invention, there isprovided an isolated nucleic acid comprising a nucleic acid sequenceselected from the group consisting of:

nucleic acid sequences corresponding to the genomic sequence of the FDgene including introns and exons as shown in FIGS. 6-1 to 6-42;

nucleic acid sequences corresponding to the nucleic acid sequence of theFD gene as shown in FIGS. 6-1 to 6-42, wherein the thymine nucleotide atposition 34,201 is replaced by a cytosine nucleotide;

nucleic acid sequences corresponding to the nucleic acid sequence of theFD gene as shown in FIGS. 6-1 to 6-42, wherein the guanine nucleotide atposition 33,714 is replaced by a cytosine nucleotide;

nucleic acid sequences corresponding to the nucleic acid sequence of theFD gene as shown in FIGS. 6-1 to 6-42, wherein the thymine nucleotide atposition 34,201 is replaced by a cytosine nucleotide and the guaninenucleotide at position 33,714 is replaced by a cytosine nucleotide;

nucleic acid sequences corresponding to the cDNA sequence including thecoding sequence of the FD gene as shown in FIGS. 7A-7C;

nucleic acid sequences corresponding to the cDNA sequence shown in FIGS.7A-7C, wherein the arginine at position 696 is replaced by a proline.

In accordance with another aspect of the present invention, there isprovided a nucleic acid probe, comprising a nucleotide sequencecorresponding to a portion of a nucleic acid as set forth in any one ofthe foregoing nucleic acid sequences.

In accordance with another aspect of the present invention, there isprovided a cloning vector comprising a coding sequence of a nucleic acidas set forth above and a replicon operative in a host cell for thevector.

In accordance with another aspect of the present invention, there isprovided an expression vector comprising a coding sequence of a nucleicacid set forth above operably linked with a promoter sequence capable ofdirecting expression of the coding sequence in host cells for thevector.

In accordance with another aspect of the present invention, there isprovided host cells transformed with a vector as set forth above.

In accordance with another aspect of the present invention, there isprovided a method of producing a mutant FD polypeptide comprising:transforming host cells with a vector capable of expressing apolypeptide from a nucleic acid sequence as set forth above; culturingthe cells under conditions suitable for production of the polypeptide;and recovering the polypeptide.

In accordance with another aspect of the present invention, there isprovided a peptide product selected from the group consisting of: apolypeptide having an amino acid sequence corresponding to the aminoacid sequence shown in FIG. 8; a polypeptide containing a mutation inthe amino acid sequence shown in FIG. 8, wherein the arginine atposition 696 is replaced with a proline; a peptide comprising at least 6amino acid residues corresponding to the amino acid sequence shown inFIG. 8, and a peptide comprising at least 6 amino acid residuescorresponding to a mutated form of the amino acid sequence shown in FIG.8. In one embodiment, the peptide is labeled. In another embodiment, thepeptide is a fusion protein.

In accordance with another aspect of the present invention, there isprovided a use of a peptide as set forth above as an immunogen for theproduction of antibodies. In one embodiment, there is provided anantibody produced in such application. In one embodiment, the antibodyis labeled. In another embodiment, the antibody is bound to a solidsupport.

In accordance with another aspect of the present invention, there isprovided a method to determine the presence or absence of the familialdysautonomia (FD) gene mutation in an individual, comprising: isolatinggenomic DNA, cDNA, or RNA from a potential FD disease carrier orpatient; and assessing the DNA for the presence or absence of anFD-associated allele, wherein said FD-associated allele is the FDIand/or FD2 mutation wherein, the absence of either FD-associated alleleindicates the absence of the FD gene mutation in the genome of theindividual and the presence of the allele indicates that the individualis either affected with FD or a heterozygote carrier.

In one embodiment, the assessing step is performed by a process whichcomprises subjecting the DNA to amplification using oligonucleotideprimers flanking the FD1 mutation and the FD2 mutation. In anotherembodiment, the assessing step further comprises an allele-specificoligonucleotide hybridization assay.

In another embodiment, DNA is amplified using the followingoligonucleotide primers: 5′-GCCAGTGTTTTTGCCTGAG-3′;5′-CGGATTGTCACTGTTGTGC-3′; 5′-GACTGCTCTCATAGCATCGC-3′. In anotherembodiment, the assessing step further comprises an allele-specificoligonucleotide hybridization assay. In another embodiment, theallele-specific oligonucleotide hybridization assay is accomplishedusing the following oligonucleotides: 5′-AAGTAAG(T/C)GCCATTG-3′ and5′-GGTTCAC(G/C)GATTGTC. In yet another embodiment, neuronal tissue froman individual is screened for the presence of truncated IKBKAP mRNA orpeptides, wherein the presence of said truncated mRNA or peptidesindicates that said individual possesses the FD1 and/or FD2 mutation inthe IKBKAP gene.

In accordance with another aspect of the present invention, there isprovided an animal model for familial dysautonomia (FD), comprising amammal possessing a mutant or knock-out or knock-in FD gene. In anotherembodiment, there is provided a method of producing a transgenic animalexpressing a mutant IKAP mRNA comprising:

(a) introducing into an embryonal cell of an animal a promoter operablylinked to the nucleotide sequence containing a mutation associated withFD;

(b) transplanting the transgenic embryonal target cell formed therebyinto a recipient female parent; and

(c) identifying at least one offspring containing said nucleotidesequence in said offspring's genome.

In accordance with another aspect of the present invention, there isprovided a method for screening potential therapeutic agents foractivity, in connection with FD, comprising: providing a screening toolselected from the group consisting of a cell line, and a mammalcontaining or expressing a defective FD gene or gene product; contactingthe screening tool with the potential therapeutic agent; and assayingthe screening tool for an activity.

In accordance with another aspect of the present invention, there isprovided a method for treating familial dysautonomia (FD) by genetherapy using recombinant DNA technology to deliver the normal form ofthe ED gene into patient cells or vectors which will supply the patientwith gene product in vivo.

In another embodiment, there is provided a method for treating familialdysautonomia (FD), comprising: providing an antibody directed against anFD protein sequence or peptide product; and delivering the antibody toaffected tissues or cells in a patient having FD.

In accordance with another aspect of the present invention, there isprovided kits for carrying out the methods of the invention. These kitsinclude nucleic acids, polypeptides and antibodies of the presentinvention. In another embodiment the kit for detecting FD mutations willalso contain genetic tests for diagnosing additional genetic diseases,such as Canavan's disease, Tay-Sachs disease, Goucher disease, CysticFibrosis, Fanconi anemia, and Bloom syndrome.

It will be appreciated by a skilled worker in the art that theidentification of the genetic defect in a genetic disease, coupled withthe provision of the DNA sequences of both normal and disease-causingalleles, provides the full scope of diagnostic and therapeutic aspectsof such an invention as can be envisaged using current technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Genomic structure of IKBKAP. The figure illustrates theorientation and placement of the 37 exons within a 68 kb genomic regionof chromosome 9q31. The primers used for analysis of the splice defectare indicated as 18F (exon 18), 19F (exon 19) and 23R (exon 23).Asterick indicates the locations of the two mutations identified; themutation associated with the major AJ haplotype is located at by 6 ofintron 20, whereas the mutation association with the minor AJ haplotypeis located at by 73 of exon 19. The 4.8 and 5.9 designations at exon 37indicate the lengths of the two IKBKAP messages that differ only in thelength of their 3′ UTRs.

FIGS. 2A-2C. Demonstration of mutations in IKBKAP. FIG. 2A shows theantisense sequence of the T→C mutation (shown by arrows adjacent to theG and A lanes) at by 6 of intron 20 that is associated with the major FDhaplotype. Lanes 1 and 2 are FD patients homozygous for the majorhaplotype (homozygous GG), lane 3 is an FD patient heterozygous for themajor haplotype and minor haplotype 2 (heterozygous GA), lane 4 is an FDpatient heterozygous for the major haplotype and minor haplotype 3(heterozygous GA), and lanes 5 and 6 are non-FD controls (homozygousAA). FIG. 2B shows heterozygosity for the G→C mutation (shown by arrowsadjacent to the G and C lanes) at by 73 of exon 19. Lane 1 is an FDhomozygous for the major haplotype (homozygous GG), lanes 2-4 are threepatients heterozygous for the major haplotype and minor haplotype 2(heterozygous GC), lane 5 is a patient heterozygous for the majorhaplotype and minor haplotype 3 (homozygous GG), and lane 6 is a non-FDcontrol (homozygous GG). FIG. 2C shows the sequence of the cDNAgenerated from the RT-PCR of a patient heterozygous for the major andminor 2 haplotypes. The arrow points to the heterozygous G→C mutation inexon 19. The boundary of exons 19 and 20 is also indicated, illustratingthat this patient expresses wild-type message that includes exon 20,despite the presence of the major mutation on one allele.

FIGS. 3A-3B. Northern blot analysis of IKBKAP. FIG. 3A is a humanmultiple tissue northern blot that was hybridized with IKBKAP exon 2 andshows the presence of two messages of 4.8 and 5.9 kb (northern blotshybridized with other IKBKAP probes yielded similar patterns). FIG. 3Bis a northern blot generated using rnRNA isolated from lymphoblast celllines: lanes 1, 2, and 5 FD patients homozygous for the major haplotype;lane 3 individual carrying two definitively non-FD chromosomes, lane 4FD patient heterozygous for the major haplotype and minor haplotype 2;lane 6 control brain RNA (Clontech). The level of expression of IKBKAPmRNA relative to (3-actin mRNA is quite variable in lymphoblasts. Weobserved no consistent increase or decrease in mRNA levels between FDpatients homozygous for the major haplotype, those heterozygous for themajor haplotype and minor haplotype 2, and non-FD individuals.

FIGS. 4A-4B: RT-PCR analysis of the exon 20 region of IKBKAP showingexpression of the wild-type message and protein in patients. FIG. 4A wasgenerated using primers 18F (exon 18) and 23R (exon 23). Lanes 1 and 2are FD patients homozygous for the major haplotype, lane 3 is an FDpatient heterozygous for the major haplotype and minor haplotype 2,lanes 4 and 5 are non-FD controls, lane 6 is a water control. FIG. 4B isa western blot generated using cytoplasmic protein isolated from patientlymphoblast cell lines and detected with a carboxyl-terminal antibody.Lanes 2, 4, 6, and 8 are patients homozygous for the major haplotype,lanes 3, 5, 7, and 9 are non-FD controls, lane 1 is a patientheterozygous for the major and minor haplotype 3, and lane 10 is apatient heterozygous for the major and minor haplotype 2 and lane 11 isa Hela cell line sample.

FIG. 5. RT-PCR analysis of the exon 20 region of IKBKAP showing variableexpression of the mutant message in FD patients. The analysis was doneusing primers 19F (exon 19) and 23F (exon 23). Lanes 1 and 2, controlfibroblasts; lanes 3, 4, and 5, FD fibroblasts homozygous for the majormutation; lanes 6 and 7 FD lymphoblasts homozygous for the majormutation, lanes 8 and 9 non-FD lymphoblasts, lane 10 FD patient brainstem, lane 11 FD patient temporal lobe (showing a faint 319 bp band andno 393 bp band), lane 12 water control. RT-PCR of control brain RNA(Clontech) showed only the 393 bp band (data not shown).

FIGS. 6-1 to 6-42. The genomic sequence for IKBKAP.

FIGS. 7A-7C—The cDNA sequence for IKBKAP.

FIG. 8—the amino acid sequence of the IKBKAP gene.

FIGS. 9A-9E—Comparison of the amino acid sequence of Ikap across severalspecies. Alignment of the amino acid sequence of Ikap (M_musculus) withthat of Homo sapiens (H_sapiens), Drosophila melanogaster (Dmelanogaster), Saccharomyces cerevisiae (S cervisiae), Arabidopsisthaliana (A_thaliana), and Caenorhabditis elegans (C_elegans).

FIG. 10—Comparison of the Novel Mouse Ikbkap Gene with Multiple SpeciesHomologs.

FIG. 11—Mouse Ikbkap Exon and Introit Boundaries.

FIG. 12—Comparison of the synthetic regions of mouse chromosome 4 (MMU4)and human chromosome 9 (HSA9q31). This diagram on the left shows thelocation of Ikbkap in relation to mapped and genetic markers (boldface).Distances are given in centimorgans. The positions of the homologousgenes that map to human chromosome 9q31 are shown on the right.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to mutations in the IKBKAP gene, which theinventors of the instant application discovered are associated withFamilial Dysautonomia. More specifically, the mutation associated withthe major haplotype of FD is a T→C change located at by 6 of intron 20in the IKBKAP gene as shown in FIG. 1. This mutation can result inskipping of exon 20 in the rnRNA from FD patients, although theycontinue to express varying levels of wild-type message in a tissuespecific manner. The mutation associated with the minor haplotype is asingle G→C change at by 2397 (bp 73 of exon 19) that causes an arginineto proline missense mutation (R696P) that is predicted to disrupt apotential phosphorylation site.

These findings have direct implications for understanding the clinicalmanifestations of FD, for preventing it and potentially for treating it.The IKAP protein produced from IKBKAP gene was originally isolated aspart of a large interleukin-1-inducible IKK complex and described as aregulator of kinases involved in pro-inflammatory cytokine signaling(Cohen et al. 1998). However, a recent report questioned thisconclusion, by reporting that cellular IKK complexes do not contain IKAPbased on various protein-protein interaction and functional assays.Rather, IKAP appears to be a member of a novel complex containingadditional unidentified proteins of 100, 70, 45, and 39 kDa (Krappmannet al. 2000).

IKAP is homologous to the EIp 1 protein of S. cerevisiae, which isencoded by the IKI3 locus and is required for sensitivity to pGKL killertoxin. The human and yeast proteins exhibit 29% identity and 46%similarity over their entire lengths. Yeast Elp 1 protein is part of theRNA polymerase II-associated elongator complex, which also containsElp2, a WD-40 repeat protein, and Elp3, a histone acetyltransferase(Otero et al. 1999). The human ELP3 gene encodes a 60 kDa histoneacetyltransferase that shows more than 75% identity with yeast Elp3protein, but no 60 kDa protein has been found in the humanIKAP-containing protein complex. Consequently, it is considered unlikelythat IKAP is a member of a functionally conserved mammalian elongatorcomplex (Krappmann et al. 2000). Instead, it has been reported that theprotein may play a role in general gene activation mechanisms, asoverexpression of IKAP interferes with the activity of bothNF-kB-dependent and independent reporter genes (Krappmann et al. 2000).Therefore, the FD phenotype may be caused by aberrant expression ofgenes crucial to the development of the sensory and autonomic nervoussystems, secondary to the loss of a functional IKAP protein in specifictissues.

FD is unique among Ashkenazi Jewish disorders in that one mutationaccounts for >99.5% of the disease chromosomes. As in other autosomalrecessive diseases with no phenotype in heterozygous carriers, one mighthave expected to find several different types of mutations producingcomplete inactivation of the DYS gene in the AJ population. The factthat the major FD mutation does not produce complete inactivation, butrather allows variable tissue-specific expression of IKAP, may explainthis lack of mutational diversity. Mutations causing completeinactivation of IKAP in all tissues might cause a more severe or evenlethal phenotype. Indeed, CG10535, the apparent Drosophila melanogasterhomologue of IKBKAP, maps coincident with a larval recessive lethalmutation (1(3)04629) supporting the essential nature of the protein(FlyBase). Thus, the array of mutations that can produce the FDphenotype may be limited if they must also allow expression offunctional or partially functional IKAP in some tissues to permitsurvival. With the identification of IKBKAP as DYS, it will now bepossible to test this inactivation hypothesis in a mammalian modelsystem.

Despite the overwhelming predominance of a single mutation in FDpatients, the disease phenotype is remarkably variable both within andbetween families. The nature of the major FD mutation makes it temptingto consider that this phenotypic variability might relate to thefrequency of exon 20 skipping in specific tissues and at specificdevelopmental stages, which may be governed by variations in manyfactors involved in RNA splicing. Even a small amount of normal IKAPprotein expressed in critical tissues might permit sufficient neuronalsurvival to alleviate the most severe phenotypes. This possibility issupported by the relatively mild phenotype associated with the presenceof the R696P mutation, which is predicted to permit expression of analtered full-length IKAP protein that may retain some functionalcapacity. To date, this minor FD mutation has only been seen in fourpatients heterozygous for the major mutation. Consequently, it isuncertain whether homozygotes for the R696P mutation would display anyphenotypic abnormality characteristic of FD. The single patient withminor haplotype 3 and mixed ancestry, whose mutation has yet to befound, is also a compound heterozygote with the major haplotype. Theexistence of minor haplotype 3 indicates that IKBKAP mutations will befound outside the AJ population, but like the R696P mutation, it isdifficult to predict the severity of phenotype that would result fromhomozygosity.

Since FD affects the development and maintenance of the sensory andautonomic nervous systems, the identification of IKBKAP as the DYS geneallows for further investigation of the role of IKAP and associatedproteins in the sensory and autonomic nervous systems. Of more immediatepractical importance, however, the discovery of the single base mutationthat characterizes >99.5% of FD chromosomes will permit efficient,inexpensive carrier testing in the AJ population, to guide reproductivechoices and reduce the incidence of FD. The nature of the major mutationalso offers some hope for new approaches to treatment of FD. Despite thepresence of this mutation, lymphoblastoid cells from patients arecapable of producing full-length wild-type mRNA and normal IKAP protein,while in neuronal tissue exon 20 is skipped, presumably leading to atruncated product. Investigation of the mechanism that permitslymphoblasts to be relatively insensitive to the potential effect of themutation on splicing may suggest strategies to prevent skipping of exon20 in other cell types. An effective treatment to prevent theprogressive neuronal loss of FD may be one aimed at facilitating theproduction of wild-type rnRNA from the mutant gene rather than exogenousadministration of the missing IKAP protein via gene therapy.

FD Screening

With knowledge of the primary mutation and secondary mutation of the FDgene as disclosed herein, screening for presymptomatic homozygotes,including prenatal diagnosis, and screening for heterozygous carrierscan be readily carried out.

1. Nucleic Acid Based Screening

Individuals carrying mutations in the FD gene may be detected at eitherthe DNA or RNA level using a variety of techniques that are well knownin the art. The genomic DNA used for the diagnosis may be obtained froman individual's cells, such as those present in peripheral blood, urine,saliva, bucca, surgical specimen, and autopsy specimens. The DNA may beused directly or may be amplified enzymatically in vitro through use ofPCR (Saiki et al. Science 239:487-491 (1988)) or other in vitroamplification methods such as the ligase chain reaction (LCR) (Wu andWallace Genomics 4:560-569 (1989)), strand displacement amplification(SDA) (Walker et al. PNAS USA 89:392-396 (1992)), self-sustainedsequence replication (3SR) (Fahy et al. PCR Methods Appl. 1:25-33(1992)), prior to mutation analysis in situ hybridization may also beused to detect the FD gene.

The methodology for preparing nucleic acids in a form that is suitablefor mutation detection is well known in the art. For example, suitableprobes for detecting a given mutation include the nucleotide sequence atthe mutation site and encompass a sufficient number of nucleotides toprovide a means of differentiating a normal from a mutant allele. Anyprobe or combination of probes capable of detecting any one of the FDmutations herein described are suitable for use in this invention.Examples of suitable probes include those complementary to either thecoding or noncoding strand of the DNA. Similarly, suitable PCR primersare complementary to sequences flanking the mutation site. Production ofthese primers and probes can be carried out in accordance with any oneof the many routine methods, e.g., as disclosed in Sambrook et al., andthose disclosed in WO 93/06244 for assays for Goucher disease.

Probes for use with this invention should be long enough to specificallyidentify or amplify the relevant FD mutations with sufficient accuracyto be useful in evaluating the risk of an individual to be a carrier orhaving the FD disorder. In general, suitable probes and primers willcomprise, preferably at a minimum, an oligomer of at least 16nucleotides in length. Since calculations for mammalian genomes indicatethat for an oligonucleotide 16 nucleotides in length, there is only onechance in ten that a typical cDNA library will fortuitously contain asequence that exactly matches the sequence of the nucleotide. Therefore,suitable probes and primers are preferably 18 nucleotides long, which isthe next larger oligonucleotide fully encoding an amino acid sequence(i.e., 6 amino acids in length).

By use of nucleotide and polypeptide sequences provided by thisinvention, safe, effective and accurate testing procedures are also madeavailable to identify carriers of mutant alleles of IKBKAP, as well aspre- and postnatal diagnosis of fetuses and live born patients carryingeither one or two mutant alleles. This affords potential parents theopportunity to make reproductive decisions prior to pregnancy, as wellas afterwards, e.g., if chorionic villi sampling or amniocentesis isperformed early in pregnancy. Thus, prospective parents who know thatthey are both carriers may wish to determine if their fetus will havethe disease, and may wish to terminate such a pregnancy, or to providethe physician with the opportunity to begin treatment as soon aspossible, including prenatally. In the case where such screening has notbeen performed, and therefore the carrier status of the patient is notknown, and where FD disease is part of the differential diagnosis, thepresent invention also provides a method for making the diagnosisgenetically.

Many versions of conventional genetic screening tests are known in theart. Several are disclosed in detail in WO 91/02796 for cystic fibrosis,in U.S. Pat. No. 5,217,865 for Tay-Sachs disease, in U.S. Pat. No.5,227,292 for neurofibromatosis and in WO 93/06244 for Goucher disease.Thus, in accordance with the state of the art regarding assays for suchgenetic disorders, several types of assays are conventionally preparedusing the nucleotides, polypeptides and antibodies of the presentinvention. For example: the detection of mutations in specific DNAsequences, such as the FD gene, can be accomplished by a variety ofmethods including, but not limited to,restriction-fragment-length-polymorphism detection based onallele-specific restriction-endonuclease cleavage (Kan and Dozy Lancetii:910-912 (1978)), hybridization with allele-specific oligonucleotideprobes (Wallace et al. Nucl Acids Res 6:3543-3557 (1978)), includingimmobilized oligonucleotides (Saiki et al. PNAS USA 86:6230-6234 (1989))or oligonucleotide arrays (Maskos and Southern Nucl Acids Res21:2269-2270 (1993)), allele-specific PCR (Newton et al. Nucl Acids Res17:2503-25 16 (1989)), mismatch-repair detection (MRD) (Faham and CoxGenome Res 5:474-482 (1995)), binding of MutS protein (Wagner et al.Nucl Acids Res 23:3944-3948 (1995), denaturing-gradient gelelectrophoresis (DGGE) (Fisher and Lerman et al. PNAS USA 80:1579-1583(1983)), single-strand-conformation-polymorphism detection (Orita et al.Genomics 5:874-879 (1983)), RNAase cleavage at mismatched base-pairs(Myers et al. Science 230:1242 (1985)), chemical (Cotton et al. PNAS USA85:4397-4401 (1988)) or enzymatic (Youil et al. PNAS USA 92:87-91(1995)) cleavage of heteroduplex DNA, methods based on allele specificprimer extension (Syvanen et al. Genomics 8:684-692 (1990)), genetic bitanalysis (GBA) (Nikiforov et al. Nucl Acids Res 22:4167-4175 (1994)),the oligonucleotide-ligation assay (OLA) (Landegren et al. Science241:1077 (1988)), the allele-specific ligation chain reaction (LCR)(Barrany PNAS USA 88:189-193 (1991)), gap-LCR (Abravaya et al. NuclAcids Res 23:675-682 (1995)), and radioactive and/or fluorescent DNAsequencing using standard procedures well known in the art.

As will be appreciated, the mutation analysis may also be performed onsamples of RNA by reverse transcription into cDNA therefrom.Furthermore, mutations may also be detected at the protein level using,for example, antibodies specific for the mutant and normal FD protein,respectively. It may also be possible to base an FD mutation assay onaltered cellular or subcellular localization of the mutant form of theFD protein.

2. Antibodies

Antibodies can also be used for the screening of the presence of the FDgene, the mutant FD gene, and the protein products therefrom. Inaddition, antibodies are useful in a variety of other contexts inaccordance with this invention. As will be appreciated, antibodies canbe raised against various epitopes of the FD protein. Such antibodiescan be utilized for the diagnosis of FD and, in certain applications,targeting of affected tissues.

For example, antibodies can be used to detect truncated FD protein inneuronal cells, the detection of which indicates that an individualpossesses a mutation in the IKBKAP gene.

Thus, in accordance with another aspect of the present invention a kitis provided that is suitable for use in screening and assaying for thepresence of the FD gene by an immunoassay through use of an antibodywhich specifically binds to a gene product of the FD gene in combinationwith a reagent for detecting the binding of the antibody to the geneproduct.

Antibodies raised in accordance with the invention can also be utilizedto provide extensive information on the characteristics of the proteinand of the disease process and other valuable information which includesbut is not limited to:

1. Antibodies can be used for the immunostaining of cells and tissues todetermine the precise localization of the FD protein. Immunofluorescenceand immuno-electron microscopy techniques which are well known in theart can be used for this purpose. Defects in the FD gene or in othergenes which cause an altered localization of the FD protein are expectedto be localizable by this method.

2. Antibodies to distinct isoforms of the FD protein (i.e., wild-type ormutant-specific antibodies) can be raised and used to detect thepresence or absence of the wild-type or mutant gene products byimmunoblotting (Western blotting) or other immunostaining methods. Suchantibodies can also be utilized for therapeutic applications where, forexample, binding to a mutant form of the FD protein reduces theconsequences of the mutation.

3. Antibodies can also be used as tools for affinity purification of FDprotein. Methods such as immunoprecipitation or column chromatographyusing immobilized antibodies are well known in the art and are furtherdescribed in Section (II)(B)(3), entitled “Protein Purification” herein.

4. Immunoprecipitation with specific antibodies is useful incharacterizing the biochemical properties of the FD protein.Modifications of the FD protein (i.e., phosphorylation, glycosylation,ubiquitization, and the like) can be detected through use of thismethod. Immunoprecipitation and Western blotting are also useful for theidentification of associating molecules that may be involved in themammalian elongation complex.

5. Antibodies can also be utilized in connection with the isolation andcharacterization of tissues and cells which express FD protein. Forexample, FD protein expressing cells can be isolated from peripheralblood, bone marrow, liver, and other tissues, or from cultured cells byfluorescence activated cell sorting (FACS) Harlow et al., eds.,Antibodies: A Laboratory Manual, pp. 394-395, Cold Spring Harbor Press,N.Y. (1988). Cells can be mixed with antibodies (primary antibodies)with or without conjugated dyes. If nonconjugated antibodies are used, asecond dye-conjugated antibody (secondary antibody) which binds to theprimary antibody can be added This process allows the specific stainingof cells or tissues which express the FD protein.

Antibodies against the FD protein are prepared by several methods whichinclude, but are not limited to:

1. The potentially immunogenic domains of the protein are predicted fromhydropathy and surface probability profiles. Then oligopeptides whichspan the predicted immunogenic sites are chemically synthesized. Theseoligopeptides can also be designed to contain the specific mutant aminoacids to allow the detection of and discrimination between the mutantversus wild-type gene products. Rabbits or other animals are immunizedwith the synthesized oligopeptides coupled to a carrier such as KLH toproduce anti-FD protein polyclonal antibodies. Alternatively, monoclonalantibodies can be produced against the synthesized oligopeptides usingconventional techniques that are well known in the art Harlow et al.,eds., Antibodies: A Laboratory Manual, pp. 151-154, Cold Spring HarborPress, N.Y. (1988). Both in vivo and in vitro immunization techniquescan be used. For therapeutic applications, “humanized” monoclonalantibodies having human constant and variable regions are oftenpreferred so as to minimize the immune response of a patient against theantibody. Such antibodies can be generated by immunizing transgenicanimals which contain human immunoglobulin genes. See Jakobovits et al.Ann NY Acad Sci 764:525-535 (1995).

2. Antibodies can also be raised against expressed FD protein productsfrom cells. Such expression products can include the full lengthexpression product or parts or fragments thereof. Expression can beaccomplished using conventional expression systems, such as bacterial,baculovirus, yeast, mammalian, and other overexpression systems usingconventional recombinant DNA techniques. The proteins can be expressedas fusion proteins with a histidine tag, glutathione-S-transferase, orother moieties, or as nonfused proteins. Expressed proteins can bepurified using conventional protein purification methods or affinitypurification methods that are well known in the art. Purified proteinsare used as immunogens to generate polyclonal or monoclonal antibodiesusing methods similar to those described above for the generation ofantipeptide antibodies.

In each of the techniques described above, once hybridoma cell lines areprepared, monoclonal antibodies can be made through conventionaltechniques of, for example, priming mice with pristane andinterperitoneally injecting such mice with the hybrid cells to enableharvesting of the monoclonal antibodies from ascites fluid.

In connection with synthetic and semi-synthetic antibodies, such termsare intended to cover antibody fragments, isotype switched antibodies,humanized antibodies (mouse-human, human-mouse, and the like), hybrids,antibodies having plural specificities, fully synthetic antibody-likemolecules, and the like.

3. Expression Systems

Expression systems for the FD gene product allow for the study of thefunction of the FD gene product, in either normal or wild-type formand/or mutated form. Such analyses are useful in providing insight intothe disease causing process that is derived from mutations in the gene.

“Expression systems” refer to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. In order to effect transformation, the expression system maybe included on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

In general terms, the production of a recombinant form of FD geneproduct typically involves the following:

First a DNA encoding the mature (used here to include all normal andmutant forms of the proteins) protein, the preprotein, or a fusion ofthe FD protein to an additional sequence cleavable under controlledconditions such as treatment with peptidase to give an active protein,is obtained. If the sequence is uninterrupted by introns it is suitablefor expression in any host. If there are introns, expression isobtainable in mammalian or other eukaryotic systems capable ofprocessing them. This sequence should be in excisable and recoverableform. The excised or recovered coding sequence is then placed inoperable linkage with suitable control sequences in an expressionvector. The construct is used to transform a suitable host, and thetransformed host is cultured under selective conditions to effect theproduction of the recombinant FD protein. Optionally the FD protein isisolated from the medium or from the cells and purified as described inSection entitled “Protein Purification”.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences can be obtained by preparingsuitable cDNA from cellular mRNA and manipulating the cDNA to obtain thecomplete sequence. Alternatively, genomic fragments may be obtained andused directly in appropriate hosts. The construction of expressionvectors operable in a variety of hosts are made using appropriatereplicons and control sequences, as set forth below. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, prokaryotic, yeast, insect, or mammalian cells are presentlyuseful as hosts. Prokaryotic hosts are in general the most efficient andconvenient for the production of recombinant proteins. However,eukaryotic cells, and, in particular, yeast and mammalian cells, areoften preferable because of their processing capacity andpost-translational processing of human proteins.

Prokaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asBacillus subtilis and various species of Pseudomonas or other bacterialstrains. In such prokaryotic systems, plasmid or bacteriophage vectorswhich contain origins of replication and control sequences compatiblewith the host are used. A wide variety of vectors for many prokaryotesare known (Maniatis et al. Molecular Cloning: A Laboratory Manual pp.1.3-1.11, 2.3-2.125, 3.2-3.48, 2-4.64 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982)); Sambrook et al. Molecular Cloning: ALaboratory Manual pp. 1-54 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)); Meth. Enzymology 68: 357-375 (1979); 101: 307-325(1983); 152: 673-864 (1987) (Academic Press, Orlando, Fla. Pouwells etal. Cloning Vectors: A Laboratory Manual (Elsevier, Amsterdam (1987))).Commonly used prokaryotic control sequences which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, include suchcommonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems, the tryptophan (trp) promoter system andthe lambda derived PL promoter and N-gene ribosome binding, site, whichhas become useful as a portable control cassette (U.S. Pat. No.4,711,845). However, any available promoter system compatible withprokaryotes can be used (Sambrook et al. supra. (1989); Meth. Enzymologysupra. (1979, 1983, 1987); John et al. Gene 61: 207-215 (1987).

In addition to bacteria, eukaryotic microbes, such as yeast, may also beused as hosts. Laboratory strain Saccharomyces cerevisiae or Baker'syeast, is most often used although other strains are commonly available.

Vectors employing the 2 micron origin of replication and other plasmidvectors suitable for yeast expression are known (Sambrook et al. supra.(1989); Meth. Enzymology supra. (1979, 1983, 1987); John et al. supra.(1987). Control sequences for yeast vectors include promoters for thesynthesis of glycolytic enzymes. Additional promoters known in the artinclude the promoters for 3-phosphoglycerate kinase, and those for otherglycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and enzymesresponsible for maltose and galactose utilization. See Sambrook et al.supra. (1989); Meth. Enzymology supra. John et al. supra. (1987). It isalso believed that terminator sequences at the 3′ end of the codingsequences are desirable. Such terminators are found in the 3′untranslated region following the coding sequences in yeast-derivedgenes. Many of the useful vectors contain control sequences derived fromthe enolase gene containing plasmid peno46 or the LEU2 gene obtainedfrom Yepl3, however, any vector containing a yeast compatible promoter,origin of replication, and other control sequences is suitable (Sambrooket al. supra. (1989); Meth. Enzymology supra. (1979, 1983, 1987); Johnet al. supra.

It is also, of course, possible to express genes encoding polypeptidesin eukaryotic host cell cultures derived from multicellular organisms(Kruse and Patterson Tissue Culture pp. 475-500 (Academic Press, Orlando(1973)); Meth. Enzymology 68: 357-375 (1979); Freshney Culture of AnimalCells; A Manual of Basic Techniques pp. 329-334 (2d ed., Alan R. Liss,N.Y. (1987))). Useful host cell lines include murine myelomas N51, VEROand HeT cells, SF9 or other insect cell lines, and Chinese hamster ovary(CHO) cells. Expression vectors for such cells ordinarily includepromoters and control sequences compatible with mammalian cells such as,for example, the commonly used early and later promoters from SimianVirus 40 (SV 40), or other viral promoters such as those from polyoma,adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, herpesvirus family (such as cytomegalovirus, herpes simplex virus, orEpstein-Barr virus), or immunoglobulin promoters and heat shockpromoters (Sambrook et al. supra. pp. 16.3-16.74 (1989); Meth.Enzymology 152: 684-704 (1987); John et al. supra. In addition,regulated promoters, such as metallothionine (i.e., MT-1 and MT-2),glucocorticoid, or antibiotic gene “switches” can be used.

General aspects of mammalian cell host system transformations have beendescribed by Axel (U.S. Pat. No. 4,399,216). Plant cells are also nowavailable as hosts, and control sequences compatible with plant cellssuch as the nopaline synthase promoter and polyadenylation signalsequences are available (Pouwells et al. supra. (1987); Meth Enzymology118: 627-639 (Academic Press, Orlando (1986); Gelvin et al. J. Bact.172: 1600-1608.

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells (Sambrook et al. supra. pp.16.30-16.5 (1989); Meth. Enzymology supra 68:357-375 (1979); 101:307-325 (1983); 152: 673-864 (1987). U.S. Pat. No. 4,399,216; MethEnzymology supra 118: 627-639 (1986); Gelvin et al. J. Bact. 172:1600-1608 (1990). Such techniques include, without limitation, calciumtreatment employing calcium chloride for prokaryotes or other cellswhich contain substantial cell wall barriers; infection withAgrobacterium tumefaciens for certain plant cells; calcium phosphateprecipitation, DEAE, lipid transfection systems (such as LIPOFECTIN™ andLIPOFFECTAMINE™), and electroporation methods for mammalian cellswithout cell walls, and, microprojectile bombardment for many cellsincluding, plant cells. In addition, DNA may be delivered by viraldelivery systems such as retroviruses or the herpes family,adenoviruses, baculoviruses, or semliki forest virus, as appropriate forthe species of cell line chosen.

C. Therapeutics

Identification of the FD gene and its gene product also has therapeuticimplications. Indeed, one of the major aims of this invention is thedevelopment of therapies to circumvent or overcome the defect leading toFD disease. Envisioned are pharmacological, protein replacement,antibody therapy, and gene therapy approaches. In addition thedevelopment of animal models useful for developing therapies and forunderstanding the molecular mechanisms of FD disease are envisioned.

1. Pharmacological

In the pharmacological approach, drugs which circumvent or overcome thedefective FD gene function are sought. In this approach, modulation ofFD gene function can be accomplished by agents or drugs which aredesigned to interact with different aspects of the FD protein structureor function.

Efficacy of a drug or agent, can be identified in a screening program inwhich modulation is monitored in vitro cell systems. Indeed, the presentinvention provides for host cell systems which express various mutant FDproteins (especially the T→C and G→C mutations noted in thisapplication) and are suited for use as primary screening systems.

In vivo testing of FD disease-modifying compounds is also required as aconfirmation of activity observed in the in vitro assays. Animal modelsof FD disease are envisioned and discussed in the section entitled“Animal Models”, below, in the present application.

Drugs can be designed to modulate FD gene and FD protein activity fromknowledge of the structure and function correlations of FD protein andfrom knowledge of the specific defect in various FD mutant proteins. Forthis, rational drug design by use of X-ray crystallography,computer-aided molecular modeling (CAMM), quantitative or qualitativestructure-activity relationship (QSAR), and similar technologies canfurther focus drug discovery efforts. Rational design allows predictionof protein or synthetic structures which can interact with and modifythe FD protein activity. Such structures may be synthesized chemicallyor expressed in biological systems. This approach has been reviewed inCapsey et al., Genetically Engineered Human Therapeutic Drugs, StocktonPress, New York (1988). Further, combinatorial libraries can bedesigned, synthesized and used in screening programs.

The present invention also envisions that the treatment of FD diseasecan take the form of modulation of another protein or step in thepathway in which the FD gene or its protein product participates inorder to correct the physiological abnormality.

In order to administer therapeutic agents based on, or derived from, thepresent invention, it will be appreciated that suitable carriers,excipients, and other agents may be incorporated into the formulationsto provide improved transfer, delivery, tolerance, and the like.

A multitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)),particularly Chapter 87, by Blaug, Seymour, therein. These formulationsinclude for example, powders, pastes, ointments, jelly, waxes, oils,lipids, anhydrous absorption bases, oil-in-water or water-in-oilemulsions, emulsions carbowax (polyethylene glycols of a variety ofmolecular weights), semi-solid gels, and semi-solid mixtures containingcarbowax.

Any of the foregoing formulations may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive agent in the formulation is not inactivated by the formulationand the formulation is physiologically compatible.

2. Protein Replacement Therapy

The present invention also relates to the use of polypeptide or proteinreplacement therapy for those individuals determined to have a defectiveFD gene. Treatment of FD disease could be performed by replacing thedefective FD protein with normal protein or its functional equivalent intherapeutic amounts.

FD polypeptide can be prepared for therapy by any of severalconventional procedures. First, FD protein can be produced by cloningthe FD cDNA into an appropriate expression vector, expressing the FDgene product from this vector in an in vitro expression system(cell-free or cell-based) and isolating the FD protein from the mediumor cells of the expression system. General expression vectors andsystems are well known in the art. In addition, the invention envisionsthe potential need to express a stable form of the FD protein in orderto obtain high yields and obtain a form readily amenable to intravenousadministration. Stable high yield expression of proteins have beenachieved through systems utilizing lipid-linked forms of proteins asdescribed in Wettstein et al. J Exp Med 174:219-228 (1991) and Lin etal. Science 249:677-679 (1990).

FD protein can be prepared synthetically. Alternatively, the FD proteincan be prepared from total protein samples by affinity chromatography.Sources would include tissues expressing normal FD protein, in vitrosystems (outlined above), or synthetic materials. The affinity matrixwould consist of antibodies (polyclonal or monoclonal) coupled to aninert matrix. In addition, various ligands which specifically interactwith the FD protein could be immobilized on an inert matrix. Generalmethods for preparation and use of affinity matrices are well known inthe art.

Protein replacement therapy requires that FD protein be administered inan appropriate formulation. The FD protein can be formulated inconventional ways standard to the art for the administration of proteinsubstances. Delivery may require packaging in lipid-containing vesicles(such as LIPOFECTIN™ or other cationic or anionic lipid or certainsurfactant proteins) that facilitate incorporation into the cellmembrane. The FD protein formulations can be delivered to affectedtissues by different methods depending on the affected tissue.

3. Gene Therapy

Gene therapy utilizing recombinant DNA technology to deliver the normalform, of the FD gene into patient cells or vectors which will supply thepatient with gene product in vivo is also contemplated within the scopeof the present invention. In gene therapy of FD disease, a normalversion of the FD gene is delivered to affected tissue(s) in a form andamount such that the correct gene is expressed and will preparesufficient quantities of FD protein to reverse the effects of themutated FD gene. Current approaches to gene therapy include viralvectors, cell-based delivery systems and delivery agents. Further, exvivo gene therapy could also be useful. In ex vivo gene therapy, cells(either autologous or otherwise) are transfected with the normal FD geneor a portion thereof and implanted or otherwise delivered into thepatient. Such cells thereafter express the normal FD gene product invivo and would be expected to assist a patient with FD disease inavoiding iron overload normally associated with FD disease. Ex vivo genetherapy is described in U.S. Pat. No. 5,399,346 to Anderson et al., thedisclosure of which is hereby incorporated by reference in its entirety.Approaches to gene therapy are discussed below:

a. Viral Vectors

Retroviruses are often considered the preferred vector for somatic genetherapy. They provide high efficiency infection, stable integration andstable expression (Friedman, T. Progress Toward Human Gene Therapy.Science 244:1275 (1989)). The full length FD gene cDNA can be clonedinto a retroviral vector driven by its endogenous promoter or from theretroviral LTR. Delivery of the virus could be accomplished by directimplantation of virus directly into the affected tissue.

Other delivery systems which can be utilized include adenovirus,adeno-associated virus (AAV), vaccinia virus, bovine papilloma virus ormembers of the herpes virus group such as Epstein-Barr virus. Virusescan be, and preferably are, replication deficient.

b. Non-Viral Gene Transfer

Other methods of inserting the FD gene into the appropriate tissues mayalso be productive. Many of these agents, however, are of lowerefficiency than viral vectors and would potentially require infection invitro, selection of transfectants, and reimplantation. This wouldinclude calcium phosphate, DEAE dextran, electroporation, and protoplastfusion. A particularly attractive idea is the use of liposomes (i.e.,LIPOFECTIN™), which might be possible to carry out in vivo. Syntheticcationic lipids and DNA conjugates also appear to show some promise andmay increase the efficiency and ease of carrying out this approach.

4. Animal Models

The generation of a mouse or other animal model of FD disease isimportant for both an understanding the biology of the disease but alsofor testing of potential therapies.

The present invention envisions the creation of an animal model of FDdisease by introduction of the FD disease causing mutations in a numberof species including mice, rats, pigs, and primates.

Techniques for specifically inactivating or mutating genes by homologousrecombination in embryonic stem cells (ES cells) have been described(Capecci Science 244:1288 (1989)). Animals with the inactivatedhomologous FD gene can then be used to introduce the mutant or normalhuman FD gene or for introduction of the homologous gene to that speciesand containing the T→C, G→C or other FD disease-causing mutations.Methods for these transgenic procedures are well known to those versedin the art and have been described by Murphy and Carter, Curr. Opin.Cell Biol. 4:273-279 (1992).

ILLUSTRATIVE EXAMPLES

The following examples are provided to illustrate certain aspects of thepresent invention and not intended as limiting the subject matterthereof.

Example 1

Identification of the IKBKAP gene and the mutations associated with FDwere obtained as follows:

Patient Samples

Blood samples were collected from two major sources, the DysautonomiaDiagnostic and Treatment Center at New York University Medical Centerand the Israeli Center for Familial Dysautonomia at Hadassah UniversityHospital, with approval from the institutional review boards at theseinstitutions, Massachusetts General Hospital and Harvard Medical School.Either F.A. or C.M. diagnosed all patients using established criteria.Epstein Barr virus transformed lymphoblast lines using standardconditions. Fibroblast cell lines were obtained from the Coriell CellRepositories, Camden, N.J. RNA isolated from post-mortem FD brain wasobtained from the Dysautonomia Diagnostic and Treatment Center at NYU.Genomic DNA, total RNA, and mnRNA were prepared using commercial kits(Invitrogen and Molecular Research Center, Inc). Cytoplasmic protein wasextracted from lymphoblasts as previously described (Krappmann et al.2000).

Identification of IKBKAP and Mutation Analysis

Exon trapping experiments of cosmids from a physical map of thecandidate region yielded 5 exons that were used to screen a humanfrontal cortex cDNA library. Several cDNA clones were isolated andassembled into a novel transcript encoding a 1332 AA protein that waslater identified as IKBKAP (Cohen et al. 1998). The complete 5.9 kb cDNAsequence of IKBKAP has been submitted to GenBank under accession numberAF153419. In order to screen for mutations in FD patients, totallymphoblast RNA was reverse transcribed and overlapping sections ofIKBKAP were amplified by PCR and sequenced. Evaluation of the splicingdefect was performed using the following primers: 18F:GCCAGTGTTTTTGCCTGAG; 19F: CGGATTGTCACTGTTGTGC; 23R: GACTGCTCTCATAGCATCGC(FIG. 1).

DNA Sequencing

Sequencing was performed using the AmpliCycle sequencing kit (AppliedBiosystems) or on an ABI 377 automated DNA sequencer rising the BigDyeterminator cycle sequencing kit (Applied Biosystems). The controlsequence of the candidate region was obtained by constructing subclonelibraries from BACs and sequencing using vector specific primers. The FDsequence was generated by sequencing cosmids from a patient homozygousfor the major FD haplotype using sequence specific primers.

Expression Studies

Several human multiple tissue northern blots (Clontech) were hybridizedusing the following radioactively labeled probes: IKBKAP exon 2, IKBKAPexons 18/19/20, IKBKAP exon 23, and a 400 bp fragment of the IKBKAP3′UTR immediately following the stop codon. Poly (A)⁺RNA was isolatedfrom patient and control lymphoblast lines, northern blotted, andhybridized using a probe representing the full coding sequence ofIKBKAP. Cytoplasmic protein extracted from lymphoblast cell lines waswestern blotted and detected using ECL (Amersharn) with an antibodyraised against a peptide comprising the extreme carboxyl terminus (AA1313-1332) of human IKAP, the protein encoded by IKBKAP (Krappmann etal. 2000).

To identify DYS, exon trapping and cDNA selection were used to clone andcharacterize all of the genes in the 471 kb candidate region: EPB41L8(unpublished data) or EHM2 (Shimizu et al. 2000), C9ORF4 (Chadwick etal. 1999a), C9ORF5 (Chadwick et al. 2000), CTNNAL1 (Zhang et al. 1998),a novel gene with homology to the glycine cleavage system H proteins(CG-8) (unpublished data), IKBKAP (Cohen et al. 1998), and ACTL7A andACTL7B (Chadwick et al. 1999b). As FD is a recessive disorder, theapriori expectation for the mutation was inactivation of one of thesegenes. Consequently, each of these were screened for mutations by RT-PCRof patient lymphoblast RNA and direct sequencing of all coding regions.Although many SNPs were identified, there was no evidence for ahomozygous inactivating mutation. Thus, it was concluded that themutation would be found in non-coding sequence and the control genomicsequence of the entire 471 kb candidate region was generated using BACsfrom a physical map. Direct sequence prediction using GENSCAN andcomprehensive searches of the public databases did not reveal anyadditional genes in the candidate region beyond those found by cloningmethods. However, SNPs identified during sequence analysis enabled us torefine the haplotype analysis and narrow the candidate interval to 177kb shared by the major haplotype and the previously described minorhaplotype 1 (Blumenfeld et al. 1999). This reduced interval contains 5genes, CTNNALI, CG-8, IKBKAP, ACTL7A and ACTL7B, all previously screenedby RT-PCR without yielding a coding sequence mutation. A cosmid librarywas constructed from a patient homozygous for the major haplotype,assembled the minimal coverage contig for the now reduced candidateinterval, and generated the sequence of the mutant chromosome.

Comparison of the FD and control sequences revealed 152 differences(excluding simple sequence repeat markers), which include 26 variationsin the length of dT_(n) tracts, 1 VNTR, and 125 base pair changes. Eachof the 125 base pair changes was tested in a panel of 50 individualsknown to carry two non-FD chromosomes by segregation in FD families. Ofthe 125 changes tested, only 1 was unique to patients carrying the majorFD haplotype. This T→C change is located at by 6 of intron 20 in theIKBKAP gene depicted in FIG. 1, and is demonstrated in FIG. 2A. IKAP wasoriginally identified as an IkB kinase (IKK) complex-associated proteinthat can bind both NF-kB inducing kinase (NIK) and IKKs through separatedomains and assemble them into an active kinase complex (Cohen et al.1998). Recent work, however, has shown that IKAP is not associated withIKKs and plays no specific role in cytokine-induced NF-kB signaling(Krappmann et al. 2000). Rather, IKAP was shown to be part of a novelmulti-protein complex hypothesized to play a role in generaltranscriptional regulation.

The IKBKAP gene contains 37 exons and encodes a 1332 amino acid protein.The full-length 5.9 kb cDNA (GenBank accession number AF153419) covers68 kb of genomic sequence, with the start methionine encoded in exon 2.IKBKAP was previously assigned to chromosome 9q34 (GenBank accessionnumber AF044195), but it clearly maps within the FD candidate region of9q31. Northern analysis of IKBKAP revealed two mRNAs of 4.8 and 5.9 kb(FIGS. 3a and b ). The wild-type 4.8 kb mRNA has been reportedpreviously (Cohen et al. 1998), while the second 5.9 kb message differsonly in the length of the 3′ UTR and is predicted to encode an identical150 kDa protein. As seen in FIG. 3B, the putative FD mutation does noteliminate expression of the IKBKAP mRNA in patient lymphoblasts.

A base pair change at position 6 of the splice donor site might beexpected to result in skipping of exon 20 (74 bp), causing a frameshiftand therefore producing a truncated protein. However, initial inspectionof our RT-PCR experiments in patient lymphoblast RNA using primerslocated in exons 18 and 23 (FIG. 1) showed a normal length 500 bpfragment that contained exon 20 (FIG. 4A), indicating that patientlymphoblasts express normal IKBKAP message. The Western blot shown inFIG. 4B demonstrates that full-length IKAP protein is expressed in thesepatient lymphoblasts. However, as the antibody used was directed againstthe carboxyl-terminus of IKAP it would not be expected to detect anytruncated protein should it be present. The presence of apparentlynormal IKAP in patient cells is at odds with the expectation of aninactivating mutation in this recessive disease.

In the absence of any evidence for a functional consequence of theintron 20 sequence change, the only alteration unique to FD chromosomes,additional genetic evidence was sought to support the view that itrepresents the FD mutation. The 658 FD chromosomes that carry the majorhaplotype all show the T→C change. In total, 887 chromosomes have beentested that are definitively non-FD due to their failure to cause thedisorder when present in individuals heterozygous for the major FDhaplotype. None of these non-FD chromosomes exhibits the T→C mutation,strongly indicating that it is not a rare polymorphism. The frequency ofthe mutation in random AJ chromosomes was 14/1012 (gene frequency 1/72;carrier frequency 1/36), close to the expected carrier frequency of 1/32(Maayan et al. 1987).

In view of the strong genetic evidence that this mutation must bepathogenic, it was postulated that its effect might be tissue-specific.RNA extracted from the brain stem and temporal lobe of a post-mortem FDbrain sample was therefore examined. In contrast to FD lymphoblasts,RT-PCR of the FD brain tissue RNA using primers in exons 19 and 23(expected to produce a normal product of 393 bp) revealed a 319 bpmutant product, indicating virtually complete absence of exon 20 fromthe IKBKAP mRNA (FIG. 5, lanes 10-11). As additional FD autopsy materialcould not be obtained, intensive analyses of additional lymphoblast andfibroblast cell lines were performed to determine whether exon-skippingcould be detected. Fibroblast lines from homozygous FD patients yieldedvariable results. Some primary fibroblast lines displayed approximatelyequal expression of the mutant and wild-type mRNAs while othersdisplayed primarily wild-type mRNA. In addition, extensive examinationof additional patient lymphoblast lines indicated that the mutantmessage could sometimes be detected at low levels. An example of thevariability seen in FD fibroblasts and the presence of the mutantmessage in some FD lymphoblasts is shown in FIG. 5. In fact, closere-examination of FIG. 4a shows a trace of the mutant band in 2 (lanes 1and 2) of the 3 FD samples. The absence of exon 20 in the FD brain RNAand the preponderance of wild-type mRNA in fibroblasts and lymphoblastsindicate that the major FD mutation acts by altering splicing of IKBKAPin a tissue-specific manner.

To identify the mutations associated with minor haplotypes 2 and 3,(Blumenfeld et al. 1999) we amplified each IKBKAP exon, includingadjacent intron sequence, from genomic DNA. A single G→C change at by2397 (bp 73 of exon 19) that causes an arginine to proline missensemutation (R696P) was identified in all 4 patients with minor haplotype 2(FIG. 2B). This was subsequently confirmed by RT-PCR in lymphoblast RNAas shown in FIG. 2C for a region that crosses the exon 19-20 border. ThePCR product, generated from an FD patient who is a compound heterozygotewith minor haplotype 2 and the major haplotype, clearly shows that RNAis being expressed equally from both alleles based on heterozygosity ofthe G→C point mutation in exon 19. However, the RNA from the majorhaplotype allele shows no evidence for skipping of exon 20 which wouldbe expected to produce a mixture of exon 20 and 21 sequence beginning atthe end of exon 19. This confirms our previous observation thatlymphoblasts with the major FD mutation produce a predominance of normalIKBKAP transcript.

The R696P mutation is absent from 500 non-FD chromosomes, and it hasbeen seen only once in 706 random AJ chromosomes in an individual whoalso carries the minor haplotype. This mutation is predicted to disrupta potential threonine phosphorylation site at residue 699 identified byNetphos 2.0 (Blom et al. 1999), suggesting that it may affect regulationof IKAP. Interestingly, the presence of this minor mutation isassociated with a relatively mild disease phenotype, suggesting that apartially functional IKAP protein may be expressed from this allele. Nomutation has been identified for minor haplotype 3, which represents theonly non-AJ putative FD chromosome.

Example 2 FD Diagnostic Assays

As discussed above, the allele-specific oligonucleotide (ASO)hybridization assay is highly effective for detecting single nucleotidechanges in DNA and RNA, such as the T→C or G→C mutations or sequencevariations, especially when used in conjunction with allele-specific PCRamplification. Thus, in accordance with the present invention, there isprovided an assay kit to detect mutations in the FD gene through use ofa PCR/ASO hybridization assay.

PCR Amplycation

Genomic DNA samples are placed into a reaction vessel(s) withappropriate primers, nucleotides, buffers, and salts and subjected toPCR amplification.

Suitable genomic DNA-containing samples from patients can be readilyobtained and the DNA extracted therefrom using conventional techniques.For example, DNA can be isolated and prepared in accordance with themethod described in Dracopoli, N. et al. eds. Current Protocols in HumanGenetics pp. 7.1.1-7.1.7 (J. Wiley & Sons, New York (1994)), thedisclosure of which is hereby incorporated by reference in its entirety.Most typically, a blood sample, a buccal swab, a hair folliclepreparation, or a nasal aspirate is used as a source of cells to providethe DNA.

Alternatively, RNA from an individual (i.e., freshly transcribed ormessenger RNA) can be easily utilized in accordance with the presentinvention for the detection of the FD2 mutation. Total RNA from anindividual can be isolated according to the procedure outlined inSambrook, J. et al. Molecular Cloning—A Laboratory Manual pp. 7.3-7.76(2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989)) thedisclosure of which is hereby incorporated by reference.

In a preferred embodiment, the DNA-containing sample is a blood samplefrom a patient being screened for FD.

In amplification, a solution containing the DNA sample (obtained eitherdirectly or through reverse transcription of RNA) is mixed with analiquot of each of dATP, dCTP, dGTP and dTTP (i.e., Pharmacia LKBBiotechnology, N.J.), an aliquot of each of the DNA specific PCRprimers, an aliquot of Taq polymerase (i.e., Promega, Wis.), and analiquot of PCR buffer, including MgCl₂ (i.e., Promega) to a finalvolume. Followed by pre-denaturation (i.e., at 95° C. for 7 minutes),PCR is carried out in a DNA thermal cycler (i.e., Perkin-Elmer Cetus,Conn.) with repetitive cycles of annealing, extension, and denaturation.As will be appreciated, such steps can be modified to optimize the PCRamplification for any particular reaction, however, exemplary conditionsutilized include denaturation at 95° C. for 1 minute, annealing at 55°C. for 1 minute, and extension at 72° C. for 4 minutes, respectively,for 30 cycles. Further details of the PCR technique can be found inErlich, “PCR Technology,” Stockton Press (1989) and U.S. Pat. No.4,683,202, the disclosure of which is incorporated herein by reference.

In a preferred embodiment, the amplification primers used for detectingthe T→C mutation and the G→C mutation in the FD gene are5′-GCCAGTGTTTTTGCCTGAG-3′/5′-GACTGCTCTCATAGCATCGC-3′ and5′-CGGATTGTCACTGTTGTGC-3′/5′-GACTGCTCTCATAGCATCGC-3, respectively.

Hybridization

Following PCR amplification, the PCR products are subjected to ahybridization assay using allele-specific oligonucleotides. In apreferred embodiment, the allele-specific oligonucleotides used todetect the mutatons in the FD gene are as follows:

5′-AAGTAAG(T/C)GCCATTG-3′ and 5′-GGTTCAC(G/C)GATTGTC.

In the ASO assay, when carried out in microliter plates, for example,one well is used for the determination of the presence of the normalallele and a second well is used for the determination of the presenceof the mutated allele. Thus, the results for an individual who isheterozygous for the T→C mutation (i.e. a carrier of FD) will show asignal in each of the wells, an individual who is homozygous for the T→Callele (i.e., affected with FD) will show a signal in only the C well,and an individual who does not have the FD mutation will show only onesignal in the T well.

In another embodiment, a kit for detecting the FD mutation by ASO assayis provided. In the kit, amplification primers for DNA or RNA (orgenerally primers for amplifying a sequence of genomic DNA, reversetranscription products, complementary products) including the T→Cmutated and normal alleles are provided. Allele-specificoligonucleotides are also preferably provided. The kit further includesseparate reaction wells and reagents for detecting the presence ofhomozygosity or heterozygosity for the T→C mutation.

Within the same kit, or in separate kits, oligonucleotides foramplification and detection of other differences (such as the G→Cmutation) can also be provided. If in the same kit as that used fordetection of the T→C mutation, separate wells and reagents are provided,and homozygosity and heterozygosity can similarly be determined.

In another embodiment a kit combining other diseases (i.e., Canavan's)

Example 3 FD Diagnostic: Other Nucleotide Based Assays

As will be appreciated, a variety of other nucleotide based detectiontechniques are available for the detection of mutations in samples ofRNA or DNA from patients. See, for example, the section, above, entitled“Nucleic Acid Based Screening.” Any one or any combination of suchtechniques can be used in accordance with the invention for the designof a diagnostic device and method for the screening of samples of DNA orRNA for FD gene mutations in accordance with the invention, such as themutations and sequence variants identified herein. Further, othertechniques, currently available, or developed in the future, which allowfor the specific detection of mutations and sequence variants in the FDgene are contemplated in accordance with the invention.

Through use of any such techniques, it will be appreciated that devicesand methods can be readily developed by those of ordinary skill in artto rapidly and accurately screen for mutations and sequence variants inthe FD gene in accordance with the invention.

Thus, in accordance with the invention, there is provided a nucleic acidbased test for FD gene mutations and sequence variants which comprisesproviding a sample of a patient's DNA or RNA and assessing the DNA orRNA for the presence of one or more FD gene mutations or sequencevariants. Samples of patient DNA or RNA (or genomic, transcribed,reverse transcribed, and/or complementary sequences to the FD gene) canbe readily obtained as described in Example 2. Through theidentification and characterization of the FD gene as taught anddisclosed in the present invention, one of ordinary skill in the art canreadily identify the genomic, transcribed, reverse transcribed, and/orcomplementary sequences to the FD gene sequence in a sample and readilydetect differences therein. Such differences in accordance with thepresent invention can be the T→C or G→C mutations or sequence variationsidentified and characterized in accordance herewith. Alternatively,other differences might similarly be detectable.

Kits for conducting and/or substantially automating the process ofidentification and detection of selected changes, as well as reagentsutilized in connection therewith, are therefore envisioned in accordancewith the invention of the present invention.

As discussed above, through knowledge of the gene-associated mutationsresponsible for FD disease, it is now possible to prepare transgenicanimals as models of the FD disease. Such animals are useful in bothunderstanding the mechanisms of FD disease as well as use in drugdiscovery efforts. The animals can be used in combination withcell-based or cell-free assays for drug screening programs.

Example 4 Creating Animal Models of FD

The first step in creating an animal model of FD is the identificationand cloning of homologs of the IKBKAP gene in other species.

Isolation of Mouse cDNA Clones

The human IKBKAP sequence (GenBank Accession No. ÁF153419) was used tosearch the mouse expressed sequence tag database (dbEST) using the BLASTprogram (www.ncbi.nlm.nih.gov/BLAST). A single 5′ EST from a mouse brainlibrary (GenBank Association No. AU079160) was identified that showedmarked similarity to the 5′ end of IKBKAP. The corresponding cDNA clone,MNCB-3931, was obtained from the Japanese Collection of the ResearchBioresource/National Institute of Infectious Disease. In addition, eightEST's that were similar to the 3′ end of the ORF were found to belong toUniGene cluster Mn.46573 (www.ncbi.nlm.nih.gov/Unigene). Examination ofthis cluster yielded several poly (A+)-containing clones, and weobtained the clone UI-M-CGOp-bhb-g-07-0-U1 (GenBank Accession No.BE994893) from Research Genetics.

RT-PCR Analysis

RNA (1 μg/ml from BALB/c mouse brain was obtained commercially(Clontech). Oligo-dT 15 and random hexamer primers were annealed to thetemplate at 65° C. for 10 min in the presence of 1× first-strand buffer,2 mM dNTP mix, and 4 mM DTT. The reaction mixture was incubated at 42°C. for 90 min after addition of Superscript™ II RT (200 U/ul) and Rnaseinhibitor (80 U/ul) (GIBCO).

DNA Sequencing and Analysis

DNA sequencing was performed using the AmpliCycle sequencing kit(Applied Biosystems) for the 33[P]-labeled dideoxynucleotide chaintermination reaction, using the following conditions: 30 sec at 94° C.,30 sec at 60° C., and 30 sec at 72° C. for 30 cycles. The radioactivelylabeled sequence reaction product was denatured at 95 C for 10 min andrun on a denaturing 6% polyacrylamide gel for autoradiography. Basicsequencing manipulations and alignments were carried out using a programfrom Genetics Computer Group (GCC; Madison, Wis.). The cDNA sequencegenerated throughout the experiments were aligned and assembled into a4799-bp cDNA named Ikbkap.

Isolation of Full-Length cDNA

To obtain the full-length cDNA sequence, PCR was performed on the mousecDNA template using primers designed from the sequence of the 5′- and3′-cDNA clones. The PCR conditions were as follows: 15 sec at 95° C., 30sec at 54° C. to 60° C., and 3 min at 68° C. for 9 cycles; then 15 secat 95° C., 30 sec at 54 to 60° C., and 3 min with increment of 5 sec foreach succeeding cycle at 68 C for 19 cycles, followed by 7 min at 72° C.The PCR products were electrophoresed on a 1% agarose gel stained withethidium bromide and were cleaner using a Qiaquick PCR cleaning kit(Qiagen) in the preparation for cycle sequencing. Successive primerswere designed in order to obtain the full-length Ikbkap sequence, whichwas deposited in GenBank under Accession No. AF367244.

Northern Blot Analysis

Expression of Ikbkap was examined using both mouse embryo and adultmouse multiple tissue Northern blots (Clontech). The blots were probedwith a 1045-bp PCR fragment that contains exons 2 through 11, which wasgenerated using primer 1 (5′-GGCGTCGTAGAAATTGC-3′) and primer 2(5′-GTGGTGCTGAAGGGGCAGGC-3′). The probe was radiolabeled (Sambrook etal., 1989) and was hybridized according to the manufacturer'sinstructions.

Chromosome Mapping of the Mouse Ikbkap Gene

Several of the mouse Ikbkap ESTs belonged to the Unigene clusterMn.46573, which has been mapped to chromosome 4 (UniSTS entry: 253051)between D4Mit287 and D4Mit197. To assess synteny between mousechromosome 4 and human chromosome 9, we used several resources availableat NCBI (www.nbci.nlm.nih.gov/Homology).

Determination of Genomic Structure of the Mouse Ikbkap

The 37 human IKBKAP exons were searched against the Celera database toobtain homologous mouse sequences. Approximately 130 mouse genomicfragments (500-700 bp) were obtained using the Celera Discovery Systemand Celera's associated database, and these fragments were assembledinto seven contigs. In order to assemble the complete genomic sequence,we obtained six mouse bacterial artificial chromosomes (BACs) fromResearcg Genetics after they screened an RPCI-23 mouse library using4300 bp human probe that contained exon 2. To verify that these BACclones contained the entire Ikbkap gene, we amplified fragments from the5′ and 3′ ends of the gene, as well as a fragment from the 3′ flankinggene Actl7b (Slaugenhaupt et al., 2001) We designed primers at the endsof each of the seven contigs constructed from the Celera data andgenerated PCR products from the BACs. Subsequently, we sequenced andclosed five of the gaps, with the resulting two contigs assembled anddeposited to Celera (Accession No CSNO09).

Creating a Targeting Vector

After cloning and sequencing the mouse homolog of the human IKBKAP gene,a targeting vector can then be constructed from the mouse genomic DNA.The targeting vector would consist of two approximately 3 kb genomicfragments from the mouse FD gene as 5′ and 3′ homologous arms. Thesearms would be chosen to flank a region critical to the function of theFD gene product (for example, exon 20).

In place of exon 20, negative and positive selectable markers can beplaced, for example, to abolish the activity of the FD gene. As apositive selectable marker a neo gene under control of phosphoglyceratekinase (pgk-1) promoter may be used and as a negative selectable markerthe 5′ arm of the vector can be flanked by a pgk-1 promoted herpessimplex thymidine kinase (HSV-TK) gene can be used.

The vector is then transfected into Rl ES cells and the transfectantsare subjected to positive and negative selection (i.e., G418 andgancyclovir, respectively, where neo and HSV-TK are used). PCR is thenused to screen for surviving colonies for the desired homologousrecombination events. These are confirmed by Southern blot analysis.

Subsequently, several mutant clones are picked and injected into C57BL/6blastocytes to produce high-percentage chimeric animals. The animals arethen mated to C57BL/6 females. Heterozygous offspring are then mated toproduce homozygous mutants. Such mutant offspring can then be tested forthe FD gene mutation by Southern blot analysis. In addition, theseanimals are tested by RT-PCR to assess whether the targeted homologousrecombination results in the ablation of the FD gene mRNA. These resultsare confirmed by Northern blot analysis and RNase protection assays.

Once established, the FD gene−/−mice can be studied for the developmentof FD-like disease and can also be utilized to examine which cells andtissue-types are involved in the FD disease process. The animals canalso be used to introduce the mutant or normal FD gene or for theintroduction of the homologous gene to that species (i.e., mouse) andcontaining the T→C or G→C mutations, or other disease causing mutations.Methods for the above-described transgenic procedures are well known tothose versed in the art and are described in detail by Murphy and Cartersupra (1993).

The techniques described above, can also be used to introduce the T→C orG→C mutations, or other homologous mutations in the animal, into thehomologous animal gene. As will be appreciated, similar techniques tothose described above, can be utilized for the creation of manytransgenic animal lines

To the extent that any reference (including books, articles, papers,patents, and patent applications) cited herein is not alreadyincorporated by reference, they are hereby expressly incorporated byreference in their entirety.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodification, and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice in the artto which the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth, and as fall within the scope of theinvention and the limits of the appended claims.

We claim:
 1. A kit for the detection of the FD2 mutation associated withFamilial Dysautonomia in a sample from a human subject, said kitcomprising an isolated oligonucleotide probe from the group consistingof (a) through (d) below: (a) an isolated oligonucletotide probeconsisting of at least 16 contiguous nucleotides of the portion of SEQID NO:1 from nucleotide 32,642 to nucleotide 36,846 which includesposition 33,714 of SEQ ID NO: 1 and being suitable for the detection ofthe FD2 mutation at position 33,714 of SEQ ID NO:1; (b) the complementof an isolated oligonucletotide probe consisting of at least 16contiguous nucleotides of the portion of SEQ ID NO:1 from nucleotide32,642 to nucleotide 36,846 which includes position 33,714 of SEQ ID NO:1 and being suitable for the detection of the FD2 mutation at position33,714 of SEQ ID NO: 1; (c) an isolated oligonucletotide probeconsisting of at least 16 contiguous nucleotides of the portion of SEQID NO:1 from nucleotide 32,642 to nucleotide 36,846 which includesposition 33,714 of SEQ ID NO: 1 except that the nucleotide which is atthe same position as position 33,714 of SEQ ID NO:1 is a cytosine andbeing suitable for the detection of the FD2 mutation at position 33,714of SEQ ID NO:1; and (d) the complement of an isolated oligonucletotideprobe consisting of at least 16 contiguous nucleotides of the portion ofSEQ ID NO:1 from nucleotide 32,642 to nucleotide 36,846 which includesposition 33,714 of SEQ ID NO: 1 except that the nucleotide which is atthe same position as position 33,714 of SEQ ID NO:1 is a cytosine andbeing suitable for the detection of the FD2 mutation at position 33,714of SEQ ID NO:
 1. 2. The kit of claim 1, wherein the isolatedoligonucleotide probe is 16 nucleotides.